Characterization of GH2 and GH42 β-galactosidases derived from bifidobacterial infant isolates

Abstract

Bifidobacteria are among the first and most abundant bacterial colonizers of the gastrointestinal tract of (breast-fed) healthy infants. Their success of colonising the infant gut is believed to be, at least partly, due to their ability to metabolize available carbon sources by means of secreted or intracellular glycosyl hydrolases (GHs). Among these, β-galactosidases are particularly relevant as they allow bifidobacteria to grow on β-galactosyl-linked saccharidic substrates, which are present in copious amounts in the milk-based diet of their infant host (e.g. lactose and human milk oligosaccharides). In the present study we employed an in silico analysis to identify GH family 2 and 42 β-galactosidases encoded by typical infant-associated bifidobacteria. Comparative genome analysis followed by characterisation of selected β-galactosidases revealed how these GH2 and GH42 members are distributed among these infant-associated bifidobacteria, while their hydrolytic activity towards growth substrates commonly available in the infant gut were also assessed.

Keywords: Bifidobacteria; HMOs; Infant gut microbiota; Lactose; β-Galactosidases.

Fig. 1

Comparative analysis of β-galactosidases in infant-derived bifidobacterial (sub)species. Heatmap showing presence (red) and absence (blue) of 18 clusters of β-galactosidase-encoding genes across the 34 bifidobacterial representatives of B. breve, B. bifidum, B. longum subsp. longum and B. longum subsp. infantis. A blue arrow highlights Cluster 15, which is present across members of all the four (sub)species, while blue, green and brown colours identify clusters observed only in B. bifidum, B. breve and B. longum species, respectively

Fig. 2

Phylogenomics of β-galactosidases. Neighbour-joining tree based on the alignment of 137 putative β-galactosidases identified across 34 members of infant-derived bifidobacterial (sub)species. A red circle highlights the genes used for experimental assessment in this study, while blue and orange squares identify those genes of which involvement in the assimilation of GOS and HMOs have been reported in literature. Circles on the phylogenetic tree highlight nodes with bootstrap values above 90% and dark blue circles identify the 14 phylogenetic groups defined by our analysis

Fig. 3

Distribution of β-galactosidases selected for functional assessment across the five bifidobacterial reference strains. a Similarity plot showing the percentage of similarity of the 20 selected β-galactosidases across the reference strains (cut-off of 70% of identity over 90% of protein length). For each gene the relative cluster of appartenance from comparative genomics is also indicated. b Hierarchical clustering analysis showing the co-occurrence of the 20 selected β-galactosidases and their cluster of orthology across the reference genomes and bifidobacterial (sub)species

Fig. 4

ONPG assay. Barplot showing the result of the ONPG assay performed on crude cell-free extracts of the 17 successfully cloned β-galactosidases. Crude cell-free extracts showing relative high level of enzymatic activity are indicated in red